Proopiomelanocortin (POMC) is expressed abundantly in the pituitary gland and arcuate nucleus of the hypothalamus where it undergoes tissue- specific posttranslational processing into multiple biologically active peptides including adrenocorticotrophic hormone (ACTH), beta-endorphin, beta- and gamma lipotropins, and alpha, beta-, and gamma-melanocyte stimulating hormones (MSH). Inappropriate expression of the POMC gene and accompanying secretion of POMC-derived peptides from pituitary adenomas is a hallmark of Cushing's disease. POMC peptides are pleiotropic and regulate the mammalian stress response, modulate neuroendocrine homeostasis, and affect behavior. Importantly, the POMC gene is expressed early in development in both neurons and the pituitary gland suggesting additional roles for beta-endorphin and MSHs in the ontogeny of the nervous system and the hypophysis. The long term objectives of the parent grant and this FIRCA proposal are to understand the molecular mechanisms regulating cell-specific and developmental POMC gene transcription and to determine the physiological function of beta- endorphin in brain and pituitary development. These goals will be achieved by the following four specific aims already described in the parent grant together with two new ones belonging to the FIRCA grant. 1) Production of melanotrophic cell lines derived from transgenic mice expressing a POMC-SV40 T antigen fusion gene to study POMC gene regulation. 2) Identification of POMC specific transcription factors in the pituitary using DNA/protein interaction assays on AtT20 corticotroph cells, new melanotrophs cells and pituitaries of transgenic mice. 3) Structure and function characterization of a novel Sp1-like transcription factor mediating pituitary specific expression of the POMC gene by affinity purification or cloning. 4) A targeted mutation that blocks the production of beta-endorphin will be introduced in mice by homologous recombination in embryonic stem cells and we will analyze the effects of the lack of beta-endorphin during development. In close relation, the goals stated in the FIRCA grant are: 1) Identification of a distal cis- acting element responsible to confer appropriate neuronal expression of the POMC gene using transgenic mice as an expression system and 2) Localization of an additional corticotroph-specific enhancer(s) necessary to achieve adequate and integration position independent levels of expression of the POMC gene using a combined strategy of DNase I hypersentitive sites identification in AtT20 cells and a deletional analysis of POMC flanking sequences in transgenic mice. Together, these studies will provide complementary information important to understand the possible causes and consequences of the pathophysiological expression of the POMC gene.